JP6729041B2 - Separation material and column - Google Patents

Description

The present invention relates to a separating material and a column.

Conventionally, in the case of separating and purifying a biopolymer represented by a protein, generally, an ion exchanger having a porous synthetic polymer as a matrix and an ion exchanger having a crosslinked gel of a hydrophilic natural polymer as a matrix. Etc. are used. In the case of an ion exchanger having a porous synthetic polymer as a matrix, the volume change due to the salt concentration is small, and when packed in a column and used in chromatography, the pressure resistance during liquid passage tends to be good. However, when this ion exchanger is used for separation of proteins and the like, non-specific adsorption such as irreversible adsorption based on hydrophobic interaction occurs and asymmetry of the peak occurs, or the hydrophobic interaction causes ion exchange. There is a problem that the protein adsorbed on the body cannot be recovered while being adsorbed.

On the other hand, an ion exchanger having a crosslinked gel of a hydrophilic natural polymer represented by a polysaccharide such as dextran or agarose as a matrix has an advantage that there is almost no nonspecific adsorption of proteins. However, this ion exchanger has the drawbacks that it significantly swells in an aqueous solution, the volume change due to the ionic strength of the solution and the volume change between the free acid form and the loaded form are large, and the mechanical strength is not sufficient. In particular, when the crosslinked gel is used for chromatography, there is a drawback that the pressure loss during the passage of the liquid is large and the gel is consolidated by the passage of the liquid.

In order to overcome the drawbacks of the hydrophilic natural polymer cross-linked gel, attempts have been made to combine it with a rigid substance that is the so-called "skeleton". For example, Patent Document 1 describes that a complex in which a gel such as a natural polymer gel is held in the pores of a porous polymer is used in the field of peptide synthesis. Patent Document 1 cites as an effect of the invention that by using such a complex, the load coefficient of the reactive substance can be increased and high yield synthesis can be performed. Further, in Patent Document 1, since the gel is surrounded by a hard synthetic polymer substance, there is no change in volume even when used in the form of a column bed, and there is an effect that the pressure of the flow-through passing through the column does not change. Have been described.

Patent Documents 2 and 3 disclose a separating material in which an inorganic porous material such as Celite is made to hold a xerogel such as a polysaccharide such as dextran or cellulose. A diethylaminomethyl (DEAE) group or the like is added to this gel in order to add sorption performance, and it is used for removing hemoglobin. As the effect, good liquid permeability in the column is described.

Patent Document 4 discloses an ion exchanger of a hybrid copolymer in which pores of a copolymer having a so-called macro network structure are filled with a gel of a cross-linked copolymer synthesized from a monomer. When the degree of cross-linking is low, the cross-linked copolymer gel has problems such as pressure loss and volume change.By using a hybrid copolymer, the liquid passing characteristics are improved and the pressure loss is reduced. Is improved and the leak behavior is improved.

A composite separating material has been proposed in which a crosslinked gel of a hydrophilic natural polymer having a huge network structure is filled in the pores of an organic synthetic polymer substrate (see Patent Documents 5 and 6).

In Patent Document 7, porous particles composed of glycidyl methacrylate and an acrylic crosslinked monomer are synthesized.

U.S. Pat. No. 4,965,289 U.S. Pat. No. 4,335,017 U.S. Pat. No. 4,336,161 U.S. Pat. No. 3,966,489 JP-A-1-254247 US Pat. No. 5,114,577 JP, 2009-244067, A JP-A-60-169427

Conventional separation materials have problems such as poor liquid permeability when used as a column, a large amount of nonspecific adsorption of biopolymers, and a low amount of adsorbed proteins. All of these problems are solved at a sufficient level. It's difficult.

Therefore, the present invention provides a separation material having excellent liquid permeability when used as a column, a nonspecific adsorption amount of biopolymers reduced, and a high protein adsorption amount, and a column including the separation material. To aim.

［３］上記水酸基を有するポリマが多糖類である、［１］又は［２］に記載の分離材。
［４］上記多糖類がデキストラン、アガロース、プルラン、アミロース及びキトサンからなる群から選ばれる少なくとも１種である、［３］に記載の分離材。
［５］当該分離材の細孔径分布におけるモード径が０．０５〜０．６μｍである、［１］〜［４］のいずれかに記載の分離材。
［６］上記分離材の粒径の変動係数が５〜１５％である、［１］〜［５］のいずれかに記載の分離材。
［７］上記第１のグラフト鎖のグラフト密度が０．１ｃｈａｉｎ／ｎｍ^２以上であり、前記第２のグラフト鎖のグラフト密度が０．１ｃｈａｉｎ／ｎｍ^２以下である、［１］〜［６］のいずれかに記載の分離材。
［８］上記第２のグラフト鎖が陽イオン交換基及び陰イオン交換基の少なくとも一方を有する、［１］〜［７］のいずれかに記載の分離材。
［９］［１］〜［８］のいずれかに記載の分離材を備えるカラム。 The present invention provides a separation material and a column described in [1] to [9] below.
[1] Porous polymer particles containing a polymer containing a styrenic monomer as a monomer unit, a first graft chain which is a zwitterionic polymer bonded to the surface of the porous polymer particles, and the first A separation material comprising a second graft chain, which is a polymer having a hydroxyl group, bonded to the graft chain.
[2] The separation material according to [1], wherein the first graft chain has a zwitterionic group represented by the following formula (I), (II), or (III).

[3] The separation material according to [1] or [2], wherein the polymer having a hydroxyl group is a polysaccharide.
[4] The separation material according to [3], wherein the polysaccharide is at least one selected from the group consisting of dextran, agarose, pullulan, amylose and chitosan.
[5] The separation material according to any one of [1] to [4], wherein the mode diameter in the pore size distribution of the separation material is 0.05 to 0.6 μm.
[6] The separation material according to any one of [1] to [5], wherein the coefficient of variation of the particle diameter of the separation material is 5 to 15%.
[7] The graft density of the first graft chain is 0.1 chain/nm ² or more, and the graft density of the second graft chain is 0.1 chain/nm ² or less, [1] to [6]. The separation material according to any one of 1.
[8] The separation material according to any one of [1] to [7], wherein the second graft chain has at least one of a cation exchange group and an anion exchange group.
[9] A column including the separation material according to any one of [1] to [8].

According to the present invention, it is possible to provide a separation material that has excellent liquid permeability when used as a column, has a reduced amount of nonspecific adsorption of biopolymers, and has a high protein adsorption amount. The present invention also provides a column including the separating material.

Hereinafter, preferred embodiments of the present invention will be described in detail, but the present invention is not limited to these embodiments. In the present specification, the “surface of the porous polymer particle” includes not only the outer surface of the porous polymer particle but also the surface of pores inside the porous polymer particle. Further, in the present specification, (meth)acrylic acid means acrylic acid or methacrylic acid, and the same applies to similar expressions such as (meth)acrylate.

The separating material according to the present embodiment includes a porous polymer particle containing a polymer containing a styrene-based monomer as a monomer unit, and a first graft chain which is a zwitterionic polymer bonded to the surface of the porous polymer particle. And a second graft chain which is a polymer having a hydroxyl group and is bonded to the first graft chain.

(Porous polymer particles)
The porous polymer particle of the present embodiment is a porous particle containing a polymer containing a monomer unit derived from one or more kinds of monomers. Porous polymer particles are, for example, particles obtained by polymerizing a monomer containing a porosifying agent. Porous polymer particles can be synthesized, for example, by conventional suspension polymerization or the like. The monomer is not particularly limited, but a vinyl monomer such as a styrene-based monomer can be used. That is, the porous polymer particles may contain a monomer unit derived from a styrenic monomer. The styrene-based monomer refers to a monomer having a styrene skeleton. Specific monomers include the following polyfunctional monomers and monofunctional monomers.

Examples of the styrenic polyfunctional monomer include divinyl compounds such as divinylbenzene, divinylbiphenyl, divinylnaphthalene, and divinylphenanthrene. These polyfunctional monomers may be used alone or in combination of two or more. Among the above, divinylbenzene is preferably used from the viewpoint of durability, acid resistance and alkali resistance. By using the styrene-based monomer containing divinylbenzene, the elastic modulus of the obtained porous polymer particles can be improved, and the particle destruction at the time of column packing and particle handling can be suppressed.

When the monomer contains divinylbenzene, the amount thereof is preferably 50% by mass or more, more preferably 60% by mass or more, further preferably 70% by mass or more, based on the total mass of the monomer. It is even more preferably 80% by mass or more. It is preferable that the content of divinylbenzene is 50% by mass or more, because the alkali resistance becomes better. The upper limit of the content of divinylbenzene based on the total mass of the monomers may be 100% by mass.

Examples of styrene-based monofunctional monomers include styrene, o-methylstyrene, m-methylstyrene, p-methylstyrene, α-methylstyrene, o-ethylstyrene, m-ethylstyrene, p-ethylstyrene, and 2 ,4-dimethylstyrene, pn-butylstyrene, pt-butylstyrene, pn-hexylstyrene, pn-octylstyrene, pn-nonylstyrene, pn-decylstyrene, p- Examples thereof include styrene such as n-dodecyl styrene, p-methoxy styrene, p-phenyl styrene, p-chloro styrene, and 3,4-dichloro styrene, and derivatives thereof. A styrene derivative having a functional group such as a carboxyl group, an amino group, a hydroxyl group and an aldehyde group can also be used. These may be used alone or in combination of two or more. Among the above, styrene is preferably used from the viewpoint of acid resistance and alkali resistance.

Examples of the porosifying agent include aliphatic or aromatic hydrocarbons, esters, ketones, ethers, and alcohols that are organic solvents that promote phase separation during polymerization and promote porosification of particles. .. Specific examples include toluene, xylene, cyclohexane, octane, butyl acetate, dibutyl phthalate, methyl ethyl ketone, dibutyl ether, 1-hexanol, 2-octanol, decanol, lauryl alcohol and cyclohexanol. These porosifying agents may be used alone or in combination of two or more.

The porosifying agent can be used in an amount of 0 to 300 parts by mass with respect to 100 parts by mass of the monomer. The porosity of the porous polymer particles can be controlled by the amount of the porosifying agent. Furthermore, the size and shape of the pores of the porous polymer particles can be controlled by the kind of the porosifying agent.

Water used as a solvent for the polymerization reaction can also be used as the porosifying agent. When water is used as the porosifying agent, it is possible to make the particles porous by dissolving the oil-soluble surfactant in the monomer and allowing the droplets of the monomer to absorb the water.

Examples of the oil-soluble surfactant used for porosification include, for example, sorbitan monoesters of branched C16-C24 fatty acids, chain unsaturated C16-C22 fatty acids or chain saturated C12-C14 fatty acids (for example, sorbitan monooleate). , Sorbitan monomyristate or sorbitan monoester derived from coconut fatty acid); diglycerol monoester of branched C16-C24 fatty acid, chain unsaturated C16-C22 fatty acid or chain saturated C12-C14 fatty acid (for example, diglycerol monoester). Oleates (eg diglycerol monoesters of C18:1 fatty acids), diglycerol monomyristate, diglycerol monoisostearate or diglycerol monoesters of coconut fatty acids); branched C16-C24 alcohols (eg Guerbet alcohols), Diglycerol monoaliphatic ethers of chain unsaturated C16-C22 alcohols or chain saturated C12-C14 alcohols (eg coconut fatty alcohols); and mixtures thereof.

Preferred oil-soluble surfactants include sorbitan monolaurate (eg, SPAN® 20, preferably greater than about 40% pure, more preferably greater than about 50%, most preferably greater than about 70% sorbitan. Monolaurate); sorbitan monooleate (eg, SPAN® 80, preferably about 40% pure, more preferably about 50%, most preferably greater than about 70% sorbitan monooleate); diglycerol mono Oleate (eg, diglycerol monooleate having a purity of greater than about 40%, more preferably greater than about 50%, most preferably greater than about 70%), diglycerol monoisostearate (eg, preferably about 40% pure). %, more preferably more than about 50%, most preferably more than about 70% diglycerol monoisostearate), diglycerol monomyristate (preferably more than about 40% pure, more preferably about 50%). Above, most preferably above about 70% diglycerol monomyristate), a cocoyl (eg, lauryl, myristoyl, etc.) ether of diglycerol, or mixtures thereof.

These oil-soluble surfactants are preferably used in the range of 5 to 80 parts by mass with respect to 100 parts by mass of the monomer. When the amount of the oil-soluble surfactant is 5 parts by mass or more, the stability of water droplets becomes sufficient, and it becomes difficult to form a large single hole. When the amount of the oil-soluble surfactant is 80 parts by mass or less, the shape of the porous polymer particles can be more easily maintained after the polymerization.

Examples of the aqueous medium used in the polymerization reaction include water, a mixed medium of water and a water-soluble solvent (eg, lower alcohol), and the like. The aqueous medium may contain a surfactant. As the surfactant, any of anionic, cationic, nonionic and zwitterionic surfactants can be used.

Examples of the anionic surfactant include fatty acid oils such as sodium oleate and potassium castor oil, alkyl sulfate salts such as sodium lauryl sulfate and ammonium lauryl sulfate, alkylbenzene sulfonates such as sodium dodecylbenzenesulfonate, and alkylnaphthalene sulfone. Acid salt, alkane sulfonate, dialkyl sulfosuccinate such as dioctyl sodium sulfosuccinate, alkenyl succinate (dipotassium salt), alkyl phosphate ester salt, naphthalene sulfonate formalin condensate, polyoxyethylene alkylphenyl ether sulfate ester salt , Polyoxyethylene alkyl ether sulfate such as sodium polyoxyethylene lauryl ether sulfate, and polyoxyethylene alkyl sulfate ester salt.

Examples of the cationic surfactant include alkylamine salts such as laurylamine acetate and stearylamine acetate, and quaternary ammonium salts such as lauryltrimethylammonium chloride.

Examples of the nonionic surfactant include hydrocarbon-based nonionic surfactants such as polyethylene glycol alkyl ethers, polyethylene glycol alkylaryl ethers, polyethylene glycol esters, polyethylene glycol sorbitan esters, polyalkylene glycol alkylamines or amides. Examples of the agent include polyether modified silicone nonionic surfactants such as polyethylene oxide adducts of silicon and polypropylene oxide adducts, and fluorine nonionic surfactants such as perfluoroalkyl glycols.

Examples of the zwitterionic surfactants include hydrocarbon surfactants such as lauryldimethylamine oxide, phosphoric acid ester surfactants and phosphorous acid ester surfactants.

As the surfactant, one type may be used alone, or two or more types may be used in combination. Among the above surfactants, an anionic surfactant is preferable from the viewpoint of dispersion stability during polymerization of the monomer.

Examples of the polymerization initiator added as necessary include benzoyl peroxide, lauroyl peroxide, benzoyl orthochloroperoxide, benzoyl orthomethoxyperoxide, 3,5,5-trimethylhexanoyl peroxide, and tert-butylperoxide. Organic peroxides such as oxy-2-ethylhexanoate and di-tert-butyl peroxide; 2,2′-azobisisobutyronitrile, 1,1′-azobiscyclohexanecarbonitrile, 2,2′ -Azo-based compounds such as azobis(2,4-dimethylvaleronitrile). The polymerization initiator can be used in the range of 0.1 to 7.0 parts by mass with respect to 100 parts by mass of the monomer.

The polymerization temperature can be appropriately selected depending on the types of the monomer and the polymerization initiator. The polymerization temperature is preferably 25 to 110°C, more preferably 50 to 100°C.

At the time of polymerization, a polymer dispersion stabilizer may be added to the emulsion in order to improve the dispersion stability of the particles.

Examples of the polymer dispersion stabilizer include polyvinyl alcohol, polycarboxylic acids, celluloses (hydroxyethyl cellulose, carboxymethyl cellulose, methyl cellulose, etc.) and polyvinylpyrrolidone, and inorganic water-soluble polymer compounds such as sodium tripolyphosphate are also used. can do. Of these, polyvinyl alcohol or polyvinyl pyrrolidone is preferable. When the polymer dispersion stabilizer is used, the addition amount of the polymer dispersion stabilizer is preferably 1 to 10 parts by mass with respect to 100 parts by mass of the monomer.

A water-soluble polymerization inhibitor such as nitrites, sulfites, hydroquinones, ascorbic acids, water-soluble vitamin Bs, citric acid, and polyphenols may be used in order to suppress the emulsion polymerization of monomers alone. ..

The average particle diameter of the porous polymer particles is preferably 500 μm or less, more preferably 300 μm or less, still more preferably 100 μm or less. The average particle size of the porous polymer particles is preferably 10 μm or more, more preferably 30 μm or more, and further preferably 50 μm or more. When the average particle diameter of the porous polymer particles is 10 μm or more, the increase in column pressure after column packing tends to be suppressed. The separation material provided with the porous polymer particles of the present embodiment is hydrodynamically and efficiently advantageous when used in chromatography, as compared with the conventional separation material obtained by pulverization.

From the viewpoint of improving liquid permeability, the coefficient of variation (C.V.) of the particle diameters of the porous polymer particles and the separating material is preferably 5 to 15%, more preferably 5 to 10%. C. of particle size V. As a method for reducing the above, monodispersion can be given by an emulsifying device such as a micro process server (manufactured by Hitachi, Ltd.).

(First graft chain)
The separating material according to the present embodiment has the first graft chain bonded to the surface of the porous polymer particles. The first graft chain is a zwitterionic polymer. By providing the separation material with the first graft chain, non-specific adsorption of biopolymer can be suppressed, and the durability of the CIP (cleaning in place) step is improved. By coating with a thin film, improvement in column pressure can be suppressed.

The zwitterionic polymer is preferably a polymer having a zwitterionic group. The zwitterionic group may be, for example, a phosphobetaine group, a carboxybetaine group, a sulfobetaine group, or the like. The zwitterionic group may be, for example, a zwitterionic group represented by the following formula (I), (II) or (III). That is, the first graft chain may have a zwitterionic group represented by the following formula (I), (II) or (III).

The first graft chain is formed by grafting a polymer having a zwitterionic group from the surface of the porous polymer particles using atom transfer radical polymerization (ATRP), which is living radical polymerization, in order to prevent nonspecific adsorption. It is desirable to do. The polymer having a zwitterionic group can be obtained, for example, by radically polymerizing a monomer including a monomer having a zwitterionic group. When the separating material has such a first graft chain, non-specific adsorption of protein can be significantly suppressed.

Examples of the monomer having a zwitterionic group include 2-(meth)acryloyloxyethylphosphorylcholine, [2-((meth)acryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, N-(meth)acryloyl. Examples thereof include oxyethyl-N,N-dimethylammonium-N-ethylcarboxylate. A (meth)acrylamide compound can also be used as the monomer having the zwitterionic functional group.

The first graft chain can be formed after introducing a graft initiation group onto the surface of the porous polymer particle. The method of introducing the graft initiation group (ATRP initiation group) in the ATRP method is not particularly limited, but when the double bond remains in the porous polymer particles, it can be reacted with bromic acid, hydrochloric acid or the like to the double bond. ATRP initiation groups can be introduced. When the surface of the porous polymer particles has a hydroxyl group, the initiation group can be easily introduced by reacting 2-bromopropionyl bromide with the porous polymer particles. In addition, as a simple method, it is also possible to introduce a starting group by forming a film on the surface of porous polymer particles using a substance obtained by reacting 2-bromopropionyl bromide with dopamine.

In the case of the ATRP method, the catalyst is not particularly limited and can be widely selected from those usually used in the ATRP method. As the catalyst, for example, a transition metal complex can be used. The transition metal complex is not particularly limited and can be widely selected. For the transition metal complex, for example, a ligand and a transition metal can be appropriately selected from the ligand group and the transition metal group exemplified below, and can be used in combination with each other. Examples of the ligand include 2,2′-bipyridyl, 4,4′-dimethyl-2,2′-dipyridyl, 4,4′-di-tert-butyl-2,2′-dipyridyl, 4,4 '-Dinonyl-2,2'-dipyridyl, N-butyl-2-pyridylmethanimine, N-octyl-2-pyridylmethanimine, N-dodecyl-N-(2-pyridyl-methylene)amine, N-octadecyl- N-(2-pyridylmethylene)amine, N,N,N',N',N'-pentamethyl-diethylenetriamine, tris(2-pyridylmethyl)amine, 1,1,4,7,10,10-hexamethyl Triethylene-tetramine, tris[2-(dimethylamino)ethylamine, 1,4,8,11-tetraazacyclotetra-decane, 1,4,8,11-tetramethyl-1-4-8-11-tetra Azacyclotetradecane, N,N,N'N'-tetrakis(2-pyridylmethyl)-ethylenediamine and the like can be mentioned.

Examples of the transition metal include CuCl, CuCl ₂ , CuBr, CuBr ₂ , TiCl ₂ , TiCl ₃ , TiCl ₄ , TiBr ₄ , FeCl ₂ , FeCl ₃ , FeBr ₂ , FeBr ₃ , CoCl ₂ , COBr ₂ , NiCl ₂ , and NiCl ₂ , NiBr _2, MoCl _3, MoCl ₅ and RuCl ₃ and the like. It is preferable to use a monovalent or divalent copper complex as the transition metal complex. The divalent copper complex is not particularly limited, but CuBr ₂ /tris[2-(dimethylamino)ethyl]amine complex or the like can be used.

When a solvent is used for the polymerization, it is not particularly limited, but a solvent used in free radical polymerization and capable of dissolving the catalyst to some extent uniformly is preferable. For example, at least one solvent selected from the group consisting of water, ethers, amides, nitriles and alcohols can be used. The ethers are not particularly limited, and examples thereof include diethyl ether, tetrahydrofuran, diphenyl ether, anisole and dimethoxybenzene. The amides are not particularly limited, and examples thereof include N,N-dimethylformamide (DMF) and N,N-dimethylacetamide. The nitriles are not particularly limited, and examples thereof include acetonitrile, propionitrile, benzonitrile and the like. The alcohols are not particularly limited, and examples thereof include methanol, ethanol, propanol, isopropanol, n-butyl alcohol, t-butyl alcohol, isoamyl alcohol and the like.

As the solvent, at least one selected from the group consisting of water, ethers, amides and alcohols is particularly preferable, and water, anisole or DMF is more preferable.

The above solvent can be used as a mixture with another solvent. Other solvents are not particularly limited, and examples thereof include solvents that are aromatic hydrocarbons and halogenated hydrocarbons. The aromatic hydrocarbon is not particularly limited, but examples thereof include benzene and toluene. The halogenated hydrocarbon is not particularly limited, but examples thereof include chlorobenzene, methylene chloride, chloroform and chlorobenzene. In this case, the amount of the above solvent is preferably not less than the molar amount of the initiator.

The graft density of the first graft chain calculated from the following formula (A) is preferably 0.1 chain/nm ² or more from the viewpoint of preventing non-specific adsorption of proteins. The graft density of the first graft chain can be, for example, 1 chain/nm ² or less. The graft density of the first graft chain can be adjusted by the surface density of the graft initiation group.
Graft density σ=graft chain amount (g/particle g)×avogadro number/(number average molecular weight Mn of graft chain×particle specific surface area (nm ² /particle g))...Equation (A)

The number average molecular weight of the first graft chain is calculated by hydrolyzing the polymer grafted on the porous polymer particles with an alkali or the like and measuring the molecular weight of the polymer in the solution by gel permeation chromatography (GPC). be able to. The amount of graft chains can be calculated by thermogravimetric analysis or by measuring the density (weight change measurement) before and after the formation of the graft chains of the porous polymer particles.

(Second graft chain)
The separating material according to the present embodiment has a second graft chain bonded to the above-mentioned first graft chain. The second graft chain is a polymer having a hydroxyl group. By having the second graft chain, the separating material can adsorb a large amount of protein. The polymer having a hydroxyl group is preferably water-soluble. The polymer to be grafted is not particularly limited as long as it has a hydroxyl group, but a polymer having many hydroxyl groups is preferable, and a polysaccharide is preferably used. The polysaccharide is preferably at least one selected from the group consisting of dextran, agarose, pullulan, amylose and chitosan. The polysaccharide preferably has a weight average molecular weight of about 10,000 to 1,000,000.

As a method for forming the second graft chain, for example, after introducing a functional group having reactivity with the second graft chain (for example, an epoxy group, a glycidyl group) into the first graft chain, A method of impregnating a solution of the polymer having a hydroxyl group into the pores of the porous polymer particle having a graft chain of 1 and reacting the polymer having a hydroxyl group with the functional group on the surface of the porous polymer particle can be mentioned. ..

As a solvent for dissolving the polymer having a hydroxyl group, any solvent can be used as long as it can dissolve the polymer, but water or alcohol is usually the most common. The concentration of the polymer dissolved in the solvent is preferably 5 to 20 (mg/ml). The impregnation can be performed, for example, by adding the porous polymer particles formed by the above method to the polymer solution having a hydroxyl group and stirring the mixture for a certain period of time. The stirring time varies depending on the surface condition of the porous polymer particles, but the impregnation for 1 to 12 hours makes the polymer concentration equilibrium with the external concentration inside the porous polymer particles. After the impregnation, the reaction is started by a reaction catalyst, heating and the like.

Specific examples of the medium in which the porous polymer particles having the first graft chain formed therein are dispersed or suspended include water, alcohol and the like. After completion of the reaction, the particles are separated by filtration, and then washed with a hydrophilic organic solvent such as water, methanol, ethanol, etc. to remove the unreacted hydrophilic polymer and suspending medium, etc. A separation material in which a second graft chain having a hydroxyl group is formed is obtained. The amount of the second graft chain can be measured by a weight loss due to thermal decomposition, a density meter or the like.

The graft density of the second graft chain is preferably 0.1 chain/nm ² or less. By setting the graft density to be equal to or lower than the above value, it becomes possible to adsorb the protein even between the molecular chains, and the adsorption amount can be improved. The graft density of the second graft chain can be, for example, 0.001 chain/nm ² or more. The graft density of the second graft chain can be adjusted by the polymer concentration in the solvent used at the time of grafting, the reaction time, and the like.

Then, the obtained separation material can be used for, for example, ion exchange purification, affinity purification or the like by introducing an ion exchange group, a ligand (protein A) or the like through the hydroxyl group on the particle surface. Examples of the method for introducing the ion exchange group include a method using an alkyl halide compound.

Examples of the halogenated alkyl compound include monohalogenocarboxylic acid and its sodium salt, primary, secondary or tertiary amine having at least one halogenated alkyl group and its hydrochloride, quaternary ammonium having a halogenated alkyl group and its Hydrochloride etc. are mentioned. Examples of the monohalogenocarboxylic acid include monohalogenoacetic acid and monohalogenopropionic acid. Examples of the tertiary amine having at least one halogenated alkyl group include diethylaminoethyl chloride and the like. The alkyl halide compound is preferably bromide or chloride. The amount of the halogenated alkyl compound used is preferably 0.2% or more with respect to the amount of the second graft chain, and 0.2% by mass with respect to the total mass of the separation material which imparts the ion-exchange groups. The above is preferable.

In order to introduce the ion exchange group, it is effective to use an organic solvent in order to accelerate the reaction. Examples of the organic solvent include alcohols such as ethanol, 1-propanol, 2-propanol, 1-butanol, isobutanol, 1-pentanol, and isopentanol.

Usually, the ion-exchange group is introduced into the polymer having a hydroxyl group existing on the surface of the separating material. The ion-exchange group is introduced, for example, by draining a wet separation material grafted with a polymer having a hydroxyl group by filtration or the like, immersing it in an alkaline aqueous solution of a predetermined concentration, and leaving it for a certain period of time, and then a water-organic solvent mixture system. Then, it can be carried out by adding the above alkyl halide compound and reacting. Examples of the alkaline aqueous solution include sodium hydroxide aqueous solution. This reaction is preferably carried out at a temperature of 40 to 90° C. under reflux for 0.5 to 12 hours. The type of alkyl halide compound used in the above reaction determines the ion exchange group to be added.

The ion exchange group may be a cation exchange group or an anion exchange group. The cation exchange group may be a weakly acidic group or a strongly acidic group. The anion exchange group may be a weakly basic group or a strongly basic group.

Examples of the method for introducing a carboxyl group, which is a weakly acidic group, as an ion-exchange group include a method in which a monohalogenocarboxylic acid such as monohalogenoacetic acid and monohalogenopropionic acid or a sodium salt thereof is reacted as the halogenated alkyl compound. The amount of the monohalogenocarboxylic acid or its sodium salt used is preferably 0.2% by mass or more based on the total mass of the separation material into which the ion exchange groups are introduced.

As a method for introducing a sulfonic acid group which is a strongly acidic group as an ion exchange group, a glycidyl compound such as epichlorohydrin is reacted with a separation material, and further a sulfite or bisulfite such as sodium sulfite or sodium bisulfite is reacted. A method of adding a separation material to a saturated aqueous solution of salt can be mentioned. The reaction is preferably carried out at 30 to 90°C for 1 to 10 hours.

As a method for providing an amino group which is a weakly basic group as an ion exchange group, at least one of the alkyl groups in the above halogenated alkyl compound is substituted with a halogenated alkyl group, mono-, di- Or a method of reacting a tri-alkylamine, a mono-alkyl-mono-alkanolamine, a di-alkyl-mono-alkanolamine, a mono-alkyl-di-alkanolamine, or the like, or at least one of the alkanol groups is a halogenated alkanol A method of reacting a mono-, di- or tri-alkanolamine, mono-alkyl-mono-alkanolamine, di-alkyl-mono-alkanolamine, mono-alkyl-di-alkanolamine, etc. Can be mentioned. The amount of these halogenated alkyl compounds used is preferably 0.2% by mass or more based on the total mass of the separating material. The reaction conditions are preferably 40 to 90° C. and 0.5 to 12 hours.

As a method of introducing a quaternary ammonium group which is a strongly basic group as an ion exchange group, first, a tertiary amino group is introduced, and the tertiary amino group is reacted with a halogenated alkyl compound such as epichlorohydrin to form a quaternary ammonium group. The method of converting into a group is mentioned. A quaternary aminohalogenide such as quaternary ammonium hydrochloride may be reacted with the separating material.

On the other hand, as another method of introducing an ion exchange group, a method of reacting 1,3-propane sultone with a separation material under an alkaline cloud atmosphere can be mentioned. It is preferable to use 0.4 mass% or more of 1,3-propane sultone based on the total mass of the separation material. The reaction is preferably performed at 0 to 90°C for 0.5 to 12 hours.

The separating material preferably has pores having a pore diameter of 0.05 to 0.6 μm, that is, those having macropores. The mode diameter of the separating material in the pore size distribution is preferably 0.05 μm to 0.6 μm. A mode diameter of 0.05 μm or more in the pore size distribution is preferable because many substances can enter the pores. When the mode diameter in the pore size distribution is 0.6 μm or less, the specific surface area increases, which is preferable. The pore size can be adjusted by the above-mentioned porosifying agent.

The specific surface area of the porous polymer particles having the first graft chain and the separating material is preferably 30 m ² /g or more. In terms of practicality, the specific surface area is more preferably 35 m ² /g or more, further preferably 40 m ² /g or more. When the specific surface area is 30 m ² /g or more, the amount of adsorbed substances to be separated tends to increase.

The ratio of the pore volume of the separating material and the porous polymer particles is preferably 30% or more and 70% or less with respect to the total volume (including the pore volume) of the separating material and the porous polymer particles, respectively. The ratio of the pore volume is more preferably 40% or more and 70% or less.

The average pore diameter of the separation material and the porous polymer particles according to the present embodiment, the mode diameter in the pore diameter distribution, the specific surface area and the porosity (pore volume) are measured by a mercury intrusion measurement device (Autopore: manufactured by Shimadzu Corporation). The values are measured as follows, and are measured as follows. About 0.05 g of the sample is added to a standard 5 ml powder cell (stem volume 0.4 ml), and the measurement is performed under the conditions of an initial pressure of 21 kPa (about 3 psia, pore diameter of about 60 μm). The mercury parameters are set to the device default of a mercury contact angle of 130 degrees and a mercury surface tension of 485 dynes/cm. Further, the respective values are calculated only within the range of the pore diameter of 0.05 to 5 μm.

The separation material according to the present embodiment has excellent non-specific adsorption due to hydrophobic interaction while maintaining excellent separation ability for separation of biopolymer such as protein, and has excellent durability. The separating material according to this embodiment is suitable for use in separating biopolymers by electrostatic interaction, affinity purification, or the like. For example, the separating material according to the present embodiment is added to a mixed solution containing a protein, and only the protein is adsorbed to the separating material by electrostatic interaction, and then the separating material is filtered from the solution to obtain a salt concentration. If it is added to an aqueous solution of high concentration, the protein adsorbed on the separation material can be easily desorbed and recovered. Further, the separation material according to the present embodiment can also be used in column chromatography. That is, the column of this embodiment includes the separation material of this embodiment.

A water-soluble substance is preferable as the biopolymer that can be separated using the separation material according to the present embodiment. Specifically, it is a blood protein such as serum albumin or immunoglobulin, a protein such as an enzyme existing in a living body, a protein bioactive substance produced by biotechnology, a DNA, a biopolymer such as a bioactive peptide. The molecular weight is preferably 2,000,000 or less, more preferably 500,000 or less. In addition, it is necessary to select the properties, conditions, etc. of the separating material according to the isoelectric point, ionization state, etc. of the protein according to a known method. Known methods include, for example, the method described in Patent Document 8 and the like.

After forming the first and second graft chains on the surface of the porous polymer particle in the present embodiment, a biopolymer such as a protein is introduced by introducing an ion exchange group, protein A and the like on the particle surface and in the pores. In the separation of the above, the characteristics which have the respective advantages of the particles composed of the natural polymer and the particles composed of the synthetic polymer are shown. In particular, the porous polymer particles, which are the skeleton of the separating material according to the present embodiment, are obtained by the above-described method, and therefore have excellent durability and alkali resistance. Further, the separation material according to the present embodiment is less likely to cause non-specific adsorption, and tends to easily desorb proteins. Furthermore, the separation material according to the present embodiment tends to have a large adsorption capacity (dynamic adsorption amount) of proteins and the like under the same flow rate.

The liquid passing rate in this specification refers to the liquid passing rate when a column is made of stainless steel or glass having a diameter of 7.8 mm x 300 mm and is filled with a separation material, and the liquid is allowed to flow. In the case of separating proteins by column chromatography, the flow rate of a solution such as a protein solution passed through a column is generally in the range of 400 cm/h or less. On the other hand, when the separation material according to the present embodiment is used, a high adsorption amount can be maintained even when the separation material for protein separation is used at a speed of 800 cm/h or more, which is faster than the conventional separation material.

The average particle size of the separating material according to this embodiment is usually preferably 10 to 300 μm. For use in preparative or industrial chromatography, it is preferably 50 to 100 μm in order to avoid an extreme increase in column internal pressure.

Furthermore, when used in column chromatography, the separation material according to the present embodiment is excellent in operability because the volume change in the column is small regardless of the property of the eluent used.

C. of the average particle size and particle size of the separating material and the porous polymer particles. V. The value can be determined by the following measuring method.
1) The particles are dispersed in water (including a dispersant such as a surfactant) to prepare a dispersion liquid containing 1% by mass of the particles.
2) Using a particle size distribution meter (Sysmex Flow, manufactured by Sysmex Co., Ltd.), the average particle size and the C. V. To measure.

In addition, in this embodiment, although the separation material of the form which introduces an ion exchange group was demonstrated, it can be used as a separation material, even if it does not introduce an ion exchange group. Such a separating material can be used, for example, in gel filtration chromatography.

Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to these examples.

(Example 1)
(Synthesis of Porous Polymer Particle 1)
In a 500 mL three-necked flask, 16 g of 96% pure divinylbenzene (DVB960) as a monomer, 6 g of SPAN80 as an oil-soluble surfactant, and 0.64 g of benzoyl peroxide as a polymerization initiator were added as a monomer phase. .. A polyvinyl alcohol (0.5 mass%) aqueous solution was prepared as a continuous phase. After emulsifying the monomer phase and the continuous phase using a micro process server, the obtained emulsion was transferred to a flask and stirred with a stirrer for about 8 hours while heating in a water bath at 80°C. The obtained particles were filtered and washed with acetone to obtain porous polymer particles 1. The particle size of the obtained porous polymer particles was measured by a flow type particle size measuring device (FPIA-3000, manufactured by Sysmex Corporation), and the average particle size (volume basis) and C.I. V. Values were calculated (Table 1).

(Formation of first graft chain)
10 g of the obtained porous polymer particles 1, 0.1 mmol of thioglycerol, and 1 mmol of azobisisobutyronitrile (AIBN) were added to 100 mL of N,N-dimethylformamide (DMF), and for 12 hours, 70 Stir at ℃. The obtained particles were washed with acetone. 10 g of the washed particles, 1 g of isobutyryl bromide, 50 g of DMF, and 0.5 g of triethylamine were stirred for 3 hours at room temperature. The obtained particles were washed with acetone to obtain particles having Br initiation groups introduced therein. 13 g of 2-(meth)acryloyloxyethylphosphorylcholine (MPC), 670 mg of copper dibromide, and 335 mg of trisdimethylaminoethylamine were added to 80 g of a 50% by volume aqueous methanol solution to prepare a solution having Br initiation groups of Nitrogen bubbling was performed by introducing 10 g of the introduced particles. Then, 200 g of ethanol in which 1190 mg of ascorbic acid was dissolved was added to the above solution and polymerized for 5 hours to form a first graft chain on the porous particle polymer. Then, 5 g of allyl glycidyl ether was added to introduce a glycidyl group at the end of the first graft chain. The particles after introduction of the glycidyl group were filtered and washed with ethanol and water. The specific surface area of the porous polymer particles having the first graft chain was measured by the mercury porosimetry method. Further, the graft amount of the first graft chain was calculated from the weight change of the obtained particles before and after the formation of the first graft chain.

The molecular weight of the graft chain was determined as follows. 1 g of the first graft chain-forming porous polymer particles was dispersed in 4 g of an aqueous sodium hydroxide solution (3N), and the polymer of the graft chain was dissolved by stirring at 25° C. for 3 hours. Was recovered. Next, the chain polymer solution was neutralized by adding 1 M hydrochloric acid until the pH of the solution became 7. The number average molecular weight of the polymer in the obtained aqueous solution was calculated by GPC. The number average molecular weight of the first graft chain was 4,500. Using the measured number average molecular weight and graft chain amount, the graft density of the first graft chain was calculated by the formula (A).

(Formation of second graft chain)
10 g of the porous polymer particles having the first graft chain obtained above were put into 100 mL of a 10 mass% aqueous solution of dextran (weight average molecular weight: 500,000) and stirred for 1 hour. Then, 100 mL of a 1 M sodium hydroxide aqueous solution was added to the above aqueous solution, and the mixture was stirred for 18 hours to carry out polymerization to form a second graft chain to obtain a separating material. The amount of the second graft chain formed on the separating material was determined by measuring the weight change of the particles before and after the formation of the second graft chain. The specific surface area of the separating material and the mode diameter in the pore size distribution were measured by the mercury porosimetry. The weight average molecular weight of dextran was regarded as the number average molecular weight, and the graft density of the second graft chain was calculated by the formula (A). The particle size of the separating material is measured by a flow type particle size measuring device (FPIA-3000, manufactured by Sysmex Corporation), and the average particle size (volume basis) and the C.I. V. Values were calculated (Table 2).

(Evaluation of non-specific adsorption ability of proteins)
0.5 g of the obtained separation material was added to 50 mL of a phosphate buffer (pH 7.4) having a BSA (bovine serum albumin) concentration of 0.5 M and a NaCl concentration of 20 mg/mL, and the mixture was stirred at room temperature for 24 hours. Then, the supernatant was collected by centrifugation, and the BSA concentration of the supernatant was measured with a spectrophotometer. The concentration of BSA was confirmed from the absorbance at 280 nm with a spectrophotometer. The amount of BSA adsorbed on the separation material was calculated from the BSA concentration of the supernatant. The amount of adsorption was 5 mg or less as “◯”, 5 to 10 mg as “Δ”, and 10 mg or more as “x”.

(Introduction of ion exchange group)
After water was removed from the obtained separation material dispersion liquid by centrifugation, 20 g of the separation material was dispersed in 100 mL of an aqueous solution in which a predetermined amount of diethylaminoethyl chloride hydrochloride was dissolved, and the mixture was stirred at 70° C. for 10 minutes. After that, 100 mL of 5M NaOH aqueous solution heated to 70° C. was added and reacted for 1 hour. After completion of the reaction, filtration and washing with water/ethanol (volume ratio 8/2) twice were performed to obtain a separation material having a diethylaminoethyl (DEAE) group as an ion exchange group. The separator having the DEAE group was used for the subsequent evaluation.

<Column characteristic evaluation>
The obtained separating material was mixed with methanol to prepare a slurry having a concentration of 30% by mass. This slurry was packed into a φ7.8×300 mm stainless steel column for 15 minutes.

(Liquid permeability evaluation)
Water was passed through the column filled with the separating material at different flow rates, the relationship between the flow rate and the column pressure was measured, and the flow rate at a column pressure of 0.3 MPa was measured. Less than 1000 cm/h was designated as “x”, 1000 cm/h or more and less than 1500 cm/h was designated as “Δ”, and 1500 cm/h or more was designated as “◯”.

(Dynamic adsorption amount)
The dynamic adsorption amount was measured as follows. 20 mmol/L Tris-hydrochloric acid buffer solution (pH 8.0) was passed through the column in 10 column volumes. Then, while measuring the BSA concentration at the column outlet by UV, a 20 mmol/L Tris-hydrochloric acid buffer solution having a BSA concentration of 2 mg/mL was caused to flow to adsorb BSA on the separation material. The liquid was allowed to flow until the BSA concentrations at the column inlet and outlet matched. Subsequently, 0.5 M NaCl Tris-hydrochloric acid buffer solution for 5 column volumes was flowed to desorb BSA from the separation material. The dynamic adsorption amount in 10% breakthrough was calculated using the following formula.
_{q 10 = c f F (t} 10 -t 0) / V B
q ₁₀ : Dynamic adsorption amount in 10% breakthrough (mg/mL wet resin)
cf: BSA concentration injected F: flow rate (mL/min)
V _B : Bed volume (mL)
t ₁₀ : Time at 10% breakthrough t ₀ : BSA infusion start time

(Cleaning in place (CIP) characteristics evaluation)
BSA was adsorbed on and desorbed from the separation material in the column in the same manner as in the evaluation of the dynamic adsorption amount, and then the column was further washed with 3 column volumes of 0.5 M NaOH aqueous solution. The cycle of BSA adsorption, desorption, and alkali washing was performed 100 times, and the reduction rate of the BSA adsorption amount in the 100th cycle relative to the BSA adsorption amount in the first cycle was recorded. When the reduction rate of the amount of BSA adsorbed was within 5%, it was marked as ⊚, when more than 5% and less than 15% was marked as “◯”, 15% or more and less than 40% was marked as “Δ”, and 40% or more was marked as “x”.

(Protein dissolution test)
The separation material was taken out from the column whose dynamic adsorption amount was evaluated and stirred in 100 g of a 0.5% by mass sodium dodecyl sulfate aqueous solution to elute the remaining adsorbed protein, and the absorbance was measured at 280 nm to remain on the carrier. The amount of protein was evaluated. Those having a residual protein amount of 5 mg or less were designated as “◯”, more than 5 mg and less than 20 mg as “Δ”, and 20 mg or more as “x”.

(Example 2)
Porous polymer particles were synthesized in the same manner as in Example 1 except that 7 g of SPAN80 was used in the synthesis of porous polymer particles (porous polymer particles 2), and a separating material was prepared and evaluated in the same manner as in Example 1. ..

(Example 3)
Porous polymer particles were synthesized in the same manner as in Example 1 except that 8 g of SPAN80 was used in the synthesis of porous polymer particles (porous polymer particles 3), and a separating material was prepared and evaluated in the same manner as in Example 1. ..

(Example 4)
A separation material was prepared in the same manner as in Example 1 except that [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (SBMA) was used as the monomer of the first graft chain instead of MPC. And evaluated.

(Example 5)
A separation material was prepared in the same manner as in Example 1 except that N-(meth)acryloyloxyethyl-N,N-dimethylammonium-N-ethylcarboxylate (CBMA) was used as the monomer of the first graft chain instead of MPC. It was produced and evaluated.

(Comparative Example 1)
A separating material was prepared and evaluated in the same manner as in Example 1 except that the second graft chain was not formed.

(Comparative example 2)
A separating material was prepared and evaluated in the same manner as in Example 1 except that hydroxyethyl methacrylate (HEMA) was used as the monomer of the first graft chain instead of MPC.

(Comparative example 3)
As the porous polymer particles 4, commercially available agarose particles (Capto DEAE, manufactured by GE Healthcare) were used as they were for evaluation.

(Comparative Example 4)
Porous polymer particles 5 were prepared in the same manner as porous polymer particles 1 except that 11.2 g of 2,3-dihydroxypropyl methacrylate, 4.8 g of ethylene glycol dimethacrylate, and 5 g of SPAN80 were used as monomers in the synthesis of porous polymer particles. Was synthesized. 4 g of the washed particles were added to 1 g of dextran (molecular weight 150,000), 6 g of sodium hydroxide 0.6 g, and a solution of 0.15 g of sodium borohydride dissolved in distilled water to impregnate the pores of the porous polymer particles. Let The obtained dextran solution-impregnated polymer was added to 1 L of a 1% by weight ethyl cellulose solution in toluene and stirred to disperse and suspend the polymer. 5 mL of epichlorohydrin was added to the obtained suspension, the temperature was raised to 50° C., and the mixture was stirred at this temperature for 6 hours to cross-link the dextran impregnated in the pores of the particles. After the completion of the reaction, the suspension was filtered to separate the produced gel-like substance from the liquid, which was sequentially washed with toluene, ethanol, and distilled water to obtain a separating material. An amino group was introduced into the obtained separation material in the same manner as in Example 1 and then evaluated.

Items marked with "*" in Tables 3 and 4 are measured after the formation of the second graft chain.

By providing the first and second graft chains on the surface of the porous polymer particles, nonspecific adsorption and protein adsorption after use could be suppressed, and the dynamic adsorption amount could be significantly improved. In addition, particles having a good linear flow velocity at a column pressure of 0.3 MPa could be obtained. On the other hand, in Comparative Example 4 obtained by impregnating the pores of the porous polymer particles with the polysaccharide, the dynamic adsorption amount was not significantly improved. It is presumed that this is because, when impregnated with a polysaccharide and cross-linked, the pores were filled and it became difficult to diffuse the protein into the particles.

Claims (6)

Porous polymer particles containing a polymer containing a styrene-based monomer as a monomer unit,
A first graft chain, which is a zwitterionic polymer, attached to the surface of the porous polymer particles;
A second graft chain, which is a polymer having a hydroxyl group, bonded to the first graft chain,
The polymer having a hydroxyl group is at least one polysaccharide selected from the group consisting of dextran, agarose, pullulan, amylose and chitosan,
The grafting density of the first graft chain is at 0.1chain / nm ² or more, the second graft density of the graft chains 0.1chain / nm ² or less der Ru separation material. The separation material according to claim 1, wherein the first graft chain has a zwitterionic group represented by the following formula (I), (II), or (III).

The separation material according to claim 1 or 2 , wherein a mode diameter in a pore size distribution of the separation material is 0.05 to 0.6 µm. The variation coefficient of the particle diameter of the separating material is 5-15%, the separation material according to any one of claims 1-3. The second graft chain having at least one of a cation exchange group and an anion-exchange group, the separation material according to any one of claims 1-4. Column comprising a separation material according to any one of claims 1-5.